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NanoHPLC-nanoESI+-MS/MS Quantitation of Bis-N7-Guanine DNA− DNA Cross-Links in Tissues of B6C3F1 Mice Exposed to subppm Levels of 1,3-Butadiene Dewakar Sangaraju,† Melissa Goggin,† Vernon Walker,§,∥ James Swenberg,‡ and Natalia Tretyakova*,† †
Department of Medicinal Chemistry and Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States ‡ University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States § Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87108, United States ∥ University of Vermont, Burlington, Vermont 05405, United States S Supporting Information *
ABSTRACT: 1,3-Butadiene (BD) is an important industrial chemical and a common environmental pollutant present in urban air. BD is classified as a human carcinogen based on epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its potent carcinogenicity in laboratory mice. A diepoxide metabolite of BD, 1,2,3,4diepoxybutane (DEB), is considered the ultimate carcinogenic species of BD due to its ability to form genotoxic DNA−DNA cross-links. We have previously employed capillary HPLC-ESI+-MS/MS (liquid chromatography-electrospray ionization tandem mass spectrometry) methods to quantify DEB-induced DNA−DNA conjugates, e.g. 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7GBD), 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and 1,N6-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′deoxyadenosine (1,N6-HMHP-dA), in tissues of laboratory mice exposed to 6.25−625 ppm BD (Goggin et al. Cancer Res. 2009, 69(6), 2479−2486). However, typical BD human exposure levels are 0.01 to 3.2 ppb in urban air and 1−2.0 ppm in an occupational setting, requiring greater detection sensitivity for these critical lesions. In the present study, a nanoHPLC-nanoESI+MS/MS method was developed for ultrasensitive, accurate, and precise quantitation of bis-N7G-BD in tissues of laboratory mice treated with low ppm and subppm concentrations of BD. The LOD value of the new method is 0.5 fmol/100 μg DNA, and the LOQ is 1.0 fmol/100 μg DNA, making it possible to quantify bis-N7G-BD adducts present at concentrations of 3 per 109 nucleotides. Bis-N7G-BD adduct amounts in liver tissues of mice exposed to 0.5, 1.0, and 1.5 ppm BD for 2 weeks were 5.7 ± 3.3, 9.2 ± 1.5, and 18.6 ± 6.9 adducts per 109 nucleotides, respectively, suggesting that bis-N7G-BD adduct formation is more efficient under low exposure conditions. To our knowledge, this is the first quantitative analysis of DEB specific DNA adducts following low ppm and subppm exposure to BD.
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INTRODUCTION 1,3-Butadiene (BD) is an important industrial chemical commonly used in rubber and plastic manufacturing1 and an environmental pollutant present in automobile exhaust2 and in cigarette smoke.3 BD is classified as a human carcinogen based on epidemiological studies in occupationally exposed workers that revealed an increased incidence of leukemia and lymphohematopoietic cancers.4−7 Furthermore, inhalation experiments with laboratory animals have shown that BD is a multisite © 2012 American Chemical Society
carcinogen, inducing lung, liver, heart, stomach, and other solid tumors in mice8 and thyroid, pancreas, uterus, testes, and other tissue tumors in rats.9 BD requires metabolic activation to DNAreactive epoxides 3,4-epoxy-1-butene (EB), 1,2,3,4-diepoxybutane (DEB), and 1,2-dihydroxy-3,4-epoxybutane (EBD).10,11 Among Received: November 18, 2011 Accepted: January 5, 2012 Published: January 5, 2012 1732
dx.doi.org/10.1021/ac203079c | Anal. Chem. 2012, 84, 1732−1739
Analytical Chemistry
Article
Scheme 1. Metabolism of 1,3-Butadiene to DEB and Its Reactions with DNA To Form Bis-N7G-BD Adducts
adducts following typical human exposures (1 ppm and lower). In the present work, we have developed and validated a nanoHPLC-nanoESI+-MS/MS method for quantitative analysis of bis-N7G-BD in vivo. The sensitivity and accuracy of the new method are as follows: LOD: 0.5 fmol/100 μg DNA, LOQ: 1.0 fmol/100 μg DNA, and accuracy: 97.1 ± 6.3% (n = 5). To our knowledge, this is the first quantitative analysis of DEB-specific DNA adducts following exposure to occupationally relevant BD concentrations (OSHA limit =1 ppm).27 Our results indicate that bis-N7G-BD formation is more efficient at low BD exposure levels (3. To evaluate the interday and intraday accuracy and precision of the new method, bis-N7G-BD (2.0 fmol) and 15N10-bis-N7GBD (50.0 fmol) were spiked into blank DNA isolated from CHL V-79 cells (100 μg). Samples were processed as described above and analyzed three times per day on three consecutive days. Method accuracy was calculated from the equation (Am/ Aa × 100%), where Am is the measured amount of bis-N7G-BD and Aa is the actual amount added. Analysis of Bis-N7G-BD in Mouse Liver Samples. DNA was extracted from livers of B6C3F1 mice exposed to 0, 0.5, 1.0, or 1.5 ppm BD by inhalation (2 weeks at 6 h/day, 5 days/
in a humidified environment containing 5% carbon dioxide and 95% air at 37 °C. Cells were subjected to serial passage at a dilution of 1:10 following brief exposure to porcine pancreatic trypsin and mechanical agitation. All experiments were performed on cells at a low passage number. DNA Isolation. DNA was isolated from mouse liver tissue using NucleoBond AXG500 anion exchange cartridges (Macherey-Nagel) according to the manufacturer’s instructions. DNA employed for method validation was isolated from CHLV79 cells by standard phenol-chloroform extraction. DNA concentrations were estimated using UV spectrophotometry. DNA purity was assessed from A260/A280 absorbance ratio, which was typically between 1.7 and 1.8. DNA amounts were determined by quantitation of dG in enzymatic hydrolysates as described below. dG Quantitation. DNA aliquots (∼10 μg) were enzymatically digested with phosphodiesterase I (6.0 mU/μg DNA), phosphodiesterase II (8.0 mU/μg DNA), DNase (3.0 U/μg DNA), and alkaline phosphatase (6.69 U/μg DNA) in 10 mM Tris-HCl/15 mM MgCl2 (pH 7.2) at 37 °C overnight. HPLCUV analysis of dG was conducted by HPLC-UV using a Zorbax SB-C8 column (4.6 × 150 mm, 5 μm, from Agilent Technologies, Palo Alto, CA) eluted with a gradient of 150 mM ammonium acetate (A) and acetonitrile (B) as described previously.29 Neutral Thermal Hydrolysis and Isolation of Bis-N7GBD Adducts. DNA samples (100 μg) were spiked with 50 fmol of S,S;R,R 15N10-bis-N7G-BD (racemic 15N10-bis-N7G-BD, internal standard for mass spectrometry) and incubated at 70 °C for 1 h to release bis-N7G-BD from the DNA backbone as a free base conjugate. Partially depurinated DNA was removed by ultrafiltration with Nanosep 10K filters (Pall Life Sciences, Ann Arbor, MI). The filtrates containing bis-N7G-BD and its internal standard were purified by offline HPLC. A Zorbax Eclipse XDBC18 (4.6 × 150 mm, 5 μm, from Agilent Technologies, Palo Alto, CA) column was eluted at 1 mL/min with a gradient of 0.4% formic acid in Milli-Q water (A) and HPLC grade acetonitrile (B). Solvent composition was maintained at 0% B for 5 min and then increased to 3% B in 10 min and further to 40% B in 5 min. The solvent composition was returned to 0% B in 5 min and maintained at 0% B for 15 min. 2′-Deoxythymidine (dT) and 2′-deoxyadenosine (dA) (12.0 nmol each) were added as retention time markers, eluting at 13.7 and 18.8 min, respectively. HPLC fractions containing bis-N7G-BD and its internal standard (14 − 18 min, about 1.25 mL) were collected, concentrated under vacuum, and dissolved in water (25 μL) for nanoHPLC-nanoESI+-MS/MS analysis. NanoHPLC-nanoESI+-MS/MS Analysis of Bis-N7G-BD. A Waters nanoAcquity UPLC system (Waters Corp., Millford, MA) interfaced to a Thermo-Finnigan TSQ Quantum UltraAM mass spectrometer (Thermo Fisher Scientific Corp., Waltham, MA) was used in all analyses. HPLC solvents were 0.01% acetic acid in LCMS grade water (A) and LC-MS grade methanol:acetonitrile (1:1) (B). Samples (8 μL) were loaded on a trapping column (Symmetry C18 nanoAcquity, 0.18 × 20 mm, Waters Corp., Millford, MA) for 1 min at 0% B at a flow rate of 10 μL/min. Chromatographic separation was achieved with a nano-HPLC column (75 μm × 200 mm) manually packed with Zorbax SB-C18, 5 μm chromatographic packing (Agilent Tech. Santa Clara, CA). The column was eluted at a flow rate of 0.4 μL/min. Solvent composition was maintained at 0% B for 1 min and then linearly increased to 10% B in 2 min, further to 50% B in 8 min, and finally to 80% B in 5 min. Solvent composition returned to 0% B for a 14 min equilibration at a flow rate of 0.4 μL/min. 1734
dx.doi.org/10.1021/ac203079c | Anal. Chem. 2012, 84, 1732−1739
Analytical Chemistry
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detection as compared to the earlier capillary HPLC-ESI-MS/ MS method that uses a flow rate of 10 μL/min.26 NanoHPLC-nanoESI+-MS/MS Method Development and Validation. MS/MS fragmentation pattern of bis-N7GBD in a triple quadrupole mass spectrometer is characterized by predominant product ion peaks at m/z 238.1 corresponding to neutral loss of guanine base from protonated molecules of the adduct [M + H − Gua]+.20,21 Another abundant fragment at m/z 152.1 corresponds to protonated guanine ions [Gua + H]+ (Figure 1). The corresponding fragments for the corresponding 15 N10 isotopically labeled internal standard are m/z 243.1 [M + H − [15N5]Gua]+ and m/z 157.1 [15N5-Gua + H]+. Our quantitative method for bis-N7G-BD is based on selected reaction monitoring of the MS/MS transitions m/z 389.1 [M + H]+ → m/z 238.1 [M + H − Gua]+ and m/z 389.1 [M + H]+ → m/z 152.1 [Gua + H]+. The corresponding transitions for the 15N10-isotopically labeled internal standard are m/z 399.1 [15N10-M + H]+ → m/z 243.1 [M + H − [15N5]Gua]+ and m/z 157.1 [15N5-Gua + H]. A sum of the two transitions is used for quantitation to improve sensitivity. To optimize analyte separation from interfering components of the biological matrix, the new method employs an offline HPLC sample cleanup. dT and dA are spiked in each sample as retention time markers, eluting at 13.7 and 18.8 min, respectively, while the retention time of bis-N7G-BD and its internal standard is ∼16 min. HPLC fractions containing bis-N7G-BD and 15N10-bis-N7G-BD (14−18 min, about 1.25 mL) were collected and concentrated for nanoLC-nanoESI+-MS/MS analysis. We have chosen nanospray LC-MS method over conventional capillary flow LC-MS because of its greater sensitivity. When HPLC flow rate is reduced to
